Targeting A-beta with evolved site-specific proteases

描述（由申请人提供）：在该提案中，我们计划开发治疗性蛋白酶制剂，旨在靶向阿尔茨海默病（AD）中假定的潜在致病机制-淀粉样蛋白-β（Ab）肽的积累-这被证明是小分子策略的困难靶标。具体而言，我们希望利用基于选择的定向进化方案，用于快速鉴定具有高选择性和催化活性的蛋白酶对抗体内的特定肽片段。最近的证据表明，淀粉样蛋白原纤维的形成，以及细胞毒性的可溶性聚集体是一个平衡控制的过程，由Ab的积累触发。此外，Ab的生物发生和清除机制的缺陷与AD的致病性有关。因此，我们假设，我们的催化剂将能够通过表现出对淀粉样蛋白生成肽的内部可降解活性来拯救Ab清除途径。具体地，我们预期单体Ab的切割将能够1）使聚集平衡向较不复杂的种类移动，以及2）以蛋白水解产物的形式原位产生有效的聚集抑制剂。因此，我们认为，Ab的位点选择性蛋白水解为开发针对这种使人衰弱的疾病的高效治疗策略提供了独特的机会，我们计划在开发这种方法时实现以下具体目标： 具体目标1：使用位点特异性蛋白酶作为起始支架，我们计划开发一种遗传选择系统，该系统能够从大量的遗传编码序列排列中识别具有针对Ab的非天然活性的独特活性物种。 具体目标二：我们将使用真实的淀粉样蛋白生成底物确认进化的蛋白酶的抗Ab活性，并进一步表征这些试剂，使用1）用于底物特异性分析的动力学分析，2）用于评估其解聚性质的双相测定，和3）用于测量其细胞保护倾向的细胞活力测定。 具体目标3：最后，我们将通过改善抗Ab蛋白酶对全身代谢的稳定性和降低其免疫原性来优化其药代动力学特性。 本研究的预期结果将是鉴定能够通过将Ab单体催化降解为非纤维蛋白原性片段和潜在的抗淀粉样蛋白片段来攻击推定AD病原体的治疗性蛋白水解剂。我们的长期目标是开发治疗策略，针对AD的潜在致病机制。

DESCRIPTION (provided by applicant): In this proposal we plan to develop therapeutic protease agents designed to target the presumed underlying pathogenic mechanism in Alzheimer's disease (AD) - accumulation of amyloid-beta (Ab) peptide - which proved to be a difficult target for small-molecule strategies. Specifically, we wish to exploit a selection-based directed evolution scheme for rapid identification of proteases with high selectivities and catalytic activities against a specific peptide fragment within Ab. Recent evidence suggests that the formation of amyloid fibrils, as well as the cytotoxic soluble aggregates is an equilibrium-controlled process, triggered by accumulation of Ab. Moreover, defects in both biogenesis and clearance mechanisms of Ab have been linked to the pathogenicity of AD. We hypothesize, therefore, that our catalytic agents will be able to rescue the Ab clearance pathway by exhibiting an internally amplifiable activity against the amyloidogenic peptide. Specifically, we expect that the cleavage of monomoric Ab will be able to 1) shift the aggregation equilibrium toward less complex species and 2) generate potent aggregation inhibitors in situ in the form of proteolysis products. We believe, therefore, that the site-selective proteolysis of Ab presents a unique opportunity for the development of a highly effective therapeutic strategy against this debilitating disease and we plan to accomplish the following Specific Aims in developing this approach: Specific Aim 1: Using a site-specific protease as a starting scaffold, we plan to develop a genetic selection system capable of identifying uniquely active species with nonnative activities against Ab from a vast collection of genetically encoded sequence permutations. Specific Aim 2: We will confirm anti-Ab activities of the evolved proteases using an authentic amyloidogenic substrate and further characterize these agents, using 1) kinetic analysis for the substrate specificity analysis, 2) biphasic assays for assessment of their deaggregation properties, and 3) cell viability assay for the measure of their cytoprotective propensity. Specific Aim 3: Finally, we will optimize the pharmacokinetic properties of the anti-Ab proteases by improving their stability to systemic metabolism and reducing their immonogenicity. The expected outcome of this research will be identification of therapeutic proteolytic agents capable of attacking putative AD pathogens through catalytic degradation of the Ab monomers into non-fibrinogenic and, potentially, anti-amyloid fragments. Our long-term goal is to develop therapeutic strategies, targeting the underlying pathogenic mechanisms in AD.